ORIGINAL Veterinary Research Forum.

2023; 14 (7) 373 - 379 Veterinary

ARTICLE doi: 10.30466/vrf.2023.562594.3631 Research
Forum
Journal Homepage: vrf.iranjournals.ir

Comparison of sperm characteristics and antioxidant and oxidant levels in bull

semen frozen with four widely used extenders
Kimia Maleki1, Esmail Ayen1*, Amir Khaki2, Ali Soleimanzadeh1
1 Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; 2 Department of Clinical Sciences, Faculty of Veterinary Medicine,
Amol University of Special Modern Technologies, Amol, Iran.

Article Info Abstract

Article history: Sperm survives for a very short time in fresh semen, and slow cooling to 5.00 ˚C kills a
large number of sperms. This study was aimed to compare the semen quality parameters
Received: 02 November 2022 and anti-oxidant levels in four extenders (manual, Triladyl, Steridyl and AndroMed).
Accepted: 06 February 2023 Semen samples were obtained from a total number of 12 dual-purpose Simmental bulls
Available online: 15 July 2023 kept in the Simmental Cattle Breeding Center for a period of 3 months using an artificial
vagina. Sperm viability, motility, abnormal morphology, plasma membrane integrity, DNA
Keywords: damage, chromatin quality, total antioxidant capacity (TAC) and lipid peroxidation were
evaluated. The highest progressive motility, viability, plasma membrane integrity, and TAC
AndroMed and the lowest levels of malondi-aldehyde in the frozen-thawed semen belonged to the
Bull semen semen group frozen with Triladyl. Parameters of motility were higher in the frozen-thawed
Cryopreservation semen with Triladyl than in other groups, indicating a significant difference from the
Steridyl manual extender. Among the extenders studied, Triladyl was the most suitable for semen
Triladyl freezing in Simmental bulls.
© 2023 Urmia University. All rights reserved.

Introduction unstable and highly reactive compounds leading to cell

damage and death.6,7
Slow cooling of undiluted semen to 5.00 ˚C kills a Egg yolk has been used as one of the main
large number of sperms, necessitating the protection components of cow ejaculate extenders.8 Low-density
during cooling to extend its lifespan.1 Freezing affects lipoproteins in egg yolk micelles and casein in milk
semen metabolism, leading to changes in metabolites stabilize the membrane and maintain sperm function
that counteract oxidative stress and regulate sperm during storage.9 The plant source of lecithin is used in
capacity and motility.2 various cold protectors based on soy lecithin Biocephos
Semen has a much higher buffering capacity than body plus, Bioxcell and AndroMed.10 The removal of animal
fluids, exerted by bicarbonate/carbon dioxide, high bio-products from the freezing protocol has been
protein, and low molecular weight compounds such as suggested due to incompatibilities in different individual
citrate, pyruvate and phosphate.3 The freeze/thaw process qualities of the particles in egg yolk.11 The ROS are among
leads to the production of reactive oxygen species (ROS) the most important free radicals.12 Mass motility and
that impairs motility, membrane strength and sperm degree of progressive sperm motility are common in
quality resulting in the early acrosomal reaction, loss of most clinical andrologists in semen analysis.13
cell contents, decreased motility and sperm fertility.4 The reason for breeding the Simmental cow is to
Under physiological conditions, the concentrations of produce the maximum milk and meat at the minimum
ROS are subtly regulated by anti-oxidants, and the result cost while using the least forage and concentrate.14
of oxidative damage is formation of malondialdehyde This project was aimed to produce the highest quality
(MDA) and other toxic by-products.5 Anti-oxidants frozen bovine sperms and find the difference between
prevent ROS formation and lipid peroxidation. Superoxide diluents for freezing bovine sperm and their effects on
dismutase, glutathione peroxidase and catalase are known sperm quality parameters and antioxidant levels of
as anti-oxidants for sperm function. Free radicals are thawed frozen semen in the freezing process.
*Correspondence:
Esmail Ayen. DVM, PhD
Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
E-mail: e.ayen@urmia.ac.ir
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as
the author of the original work is cited properly.
374 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379

Materials and Methods 1,500 mL Erlenmeyer flask. The resulting compound was
a stock solution to which 250 g of egg yolk was added.
The Animals Ethics Committee of Urmia University Steridyl (Minitube), contains the same compound as
approved the animals selected for this study (No. IR-UU- Triladyl as well as egg yolks, in 500mL bottles. The final
AEC-1861.AD.3). After obtaining the consent and diluent, 750 mL of pure double-distilled water was
permissions from Iran Simmental cow breeding and simply added to a complete package of Steridyl diluent in
sperm production center, semen samples were taken from a 1,500 mL Erlenmeyer flask. AndroMed (Minitube)
12 Simmental dual-purpose bulls (Falkfieh) kept in the consists phospholipids, Tris, citric acid, sugar, anti-
Simmental Cow Breeding and Production Center, in at oxidants, buffers, glycerol, antibiotics and the purest
least four to five stages over a period of 2 - 3 months using water in 200 and 1,000mL bottles. The current research
the synthetic vagina in 31 ejaculations. Alternative to the used 200 mL bottles of this diluent. Each pack of 200 mL
components of animal origin in semen expanders is soy of this diluent contained 11.40 mg tylosin, 57.20 mg
lecithin, a natural blend of phosphatidylcholine and gentamycin, 68.60 mg spectinomycin and 34.40 mg
several fatty acids such as stearic, oleic and palmitic.15 lincomycin. To prepare the final diluent, 800 mL of
Animals. The semen collection was carried out over a double-distilled water was simply added to a complete
4-month period from late December 2019 to mid-April package of 200 mL AndroMed diluent in a 1,000 mL
2020. Overall, 12 healthy breeding bulls of the Simmental Erlenmeyer flask.
breed aged 4 - 7 years old were used for this research. The Semen freezing. The diluted semen was packed in
samples were taken during routine weekly semen 0.50 mL straws (Minitube) with the MPP Uno automated
collection time at the Simmental Cattle Breeding Center filling and sealing machine (Minitube). Equilibration of the
(height above sea level: 47.00 m, longitude: 52° 23′57.76′′ packed semen was done by placing straws in a refrigerator
E, and latitude: 36° 30′ 18.55′′ N) between 8:00 - 12:00 set at 4.00 ˚C for 3 hr.
A.M. Animals were fed three times a day with the Sperm motility analysis. To thaw the frozen semen,
composition mentioned in Table 1. the straws were placed in a water bath adjusted at 37.00 ˚C
Semen collection and experimental groups. for 40 sec. All analyses were performed by a light
Semen samples from each bull were collected by a pre- microscope (Nikon, Tokyo, Japan) equipped with a hot
warmed artificial vagina at 46.00 ˚C in an oven (three plate and the samples were maintained in a chamber
repetitions for each cow). Measurement of the semen (sperm meter, depth 10.00 µm, surface graticule, 100 ×
concentration was carried out by the SDM photometer 0.10 mm2) at 37.00 ˚C to avoid the decline in the sperm
(Minitube, Tiefenbach, Germany) calibrated for bull motility during analysis.
sperm cell counting. Sperm viability and morphology assay. Eosin-
Extender preparation. The manual, 29.00 g of sodium Nigrosin staining was used (Fig. 1A). Eosin-Nigrosin
citrate (Sigma-Aldrich, St. Louis, USA) was dissolved in staining was used. After the sperms were placed in the
1,000 mL of double-distilled water followed by adding final diluent, 1.00 drop of sperm was mixed with 1.00
70.00 mL glycerol (Sigma-Aldrich), 250 mL egg yolk, 1.00 g drop of 2.00% Eosin and 2.00 drops of 4.00% Nigrosin
streptomycin (NASR pharmaceutical, Tehran, Iran) and with the sampler and spread. Spread on a hot, dry plate
0.70 g penicillin (NASR pharmaceutical) to 680 mL of the and immediately after (maximum 2 min) at least 200
prepared sodium citrate solution. Triladyl (Sigma-Aldrich) spermatozoa were examined under the microscope
included Tris, citric acid, glucose, buffer, glycerol, pure (Nikon). Due to the high permeability of their cell
water and antibiotics (tylosin, gentamicin, spectinomycin, membrane, dead sperms take on the color of Eosin and
and lincomycin) in 250 g bottles. Double-distilled water become red, thus, the percentage of dead spermatozoa
(750 mL) was combined to a 250g Triladyl diluent in a is obtained.16,17
Table 1. Compositions and amounts of Fleckvieh bulls’ daily meal.
Ingredient Amount (kg) Crude protein (%) NDF (%) ADF (%) Fat (%) Ash (%) Dry matter (%)
Concentrate mix * 9.00 14.68 16.80 13.10 3.20 7.40 89.20
Silage 18.00 8.50 54.50 32.70 1.80 5.70 25.00
Hay 3.00 16.80 44.70 34.60 2.50 9.70 1.88
Straw Ad libitum 3.90 70.30 45.50 1.10 9.80 94.80
Mineral supplement † Ad libitum - - - - - -
Water Ad libitum - - - - - -
* Calcium 0.74%, Phosphorus 0.53%, Sodium 0.49%, Magnesium 0.29%, Zinc 375 ppm, Manganese 381.44 ppm, Cobalt 1.01 ppm,
Selenium 2.75, and vitamin additives (vitamin A 7,500 U kg-1, vitamin D3 1,000 U kg-1, and vitamin E 10.00 mg kg-1).
† Magnesium 2.10%, Sodium 7.00%, Iron 355 mg kg-1, Zinc 1,560 mg kg-1, Copper 390 mg kg-1, Manganese 1,560 mg kg-1, Selenium 7.50
mg kg-1, Co 3.00 mg kg-1, and Iodine 15.50 mg kg-1.
NDF: neutral detergent fiber, ADF: acid detergent fiber, and Ash: total mineral content of a diet.
Source: Data calculated by Animal Nutrition Laboratory, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379
Assessment of membrane integrity. The hypo-
osmotic swelling test (HOST) was carried out following the
technique used by Revell and Mrode. Percentages of sperm
reacted in a HOST and the sperm with swollen or coiled
tails were determined for all bulls (Fig. 1B).18,19
Aniline blue staining. Aniline blue staining was used
to assess the maturity of the sperm nucleus. This analysis
was based on the fact that during spermiogenesis, protamine
was replaced by histone in the nucleus chromatin, which
was a very important replacement in sperm density and
stability. In this staining method, immature sperms turn
blue due to high histone, while adult sperms undergo less
staining (Fig. 1C).16 The sperm chromatin quality was
examined by Aniline blue, as an indicator of significant
chromatin/DNA damage. The air-dried fixed smears were
stained for 5 min in 5.00% aqueous Aniline blue solution
(5.00 g Aniline blue (Sigma-Aldrich), 4.00% acetic acid
(Sigma-Aldrich) in double-distilled water, pH = 3.50). The
percentages of spermatozoa stained with Aniline blue and
Aniline blue positive were determined by counting 400
spermatozoa per slide under a bright field microscope
(Nikon, Tokyo, Japan).20,21
Acridine orange staining. Acridine orange staining
was performed to investigate DNA damage and identify
denatured, double-stranded DNA segments in the
chromatin of the sperm at a low pH rate. In green sperms
DNA was normal and in yellow to red sperms DNA were
damaged (Fig. 1D).4,16
Fig. 1. A) Evaluation of live and abnormal sperms by Eosin-
Nigrosin staining. a: Dead sperm with red head due to plasma
membrane permeability, b: Impermeable live sperm with blue
head (200×). B) Evaluation of the sperm membrane health in
hypoosmotic solution, a: Sperm with a healthy plasma membrane
and twisted tail, b: Unhealthy sperm without reaction (400×). C)
Evaluation of excess histone by the Aniline blue method. a:
Immature sperms turn blue due to high histone, b: mature
sperms undergo less staining (400×). D) Evaluation of DNA
damage by Acridine orange staining, a: DNA damaged (yellow-
red), b: Normal sperm (green), (200×).
Total antioxidant capacity (TAC) assessment. The
TAC was measured using a kit (Naxifer™; Navand Salamat
375
Co., Urmia, Iran) in seminal plasma based on the
manufacturer’s protocols. Briefly, the diluted seminal
plasma (1:10) was mixed with stock solutions step by step.
Then, the TAC content in the final solution was detected
using a spectrophotometer (Thermo Fisher Scientific,
Waltham, USA) at 593 nm.22
Evaluation of lipid peroxidation. The products of
lipid oxidation included oxidized cholesterol MDA, lipid
hydroperoxides, and other aldehydes and ketones.23 This
method was performed according to the protocol of
Draper and Hadley.24,25 Seminal plasma MDA status was
detected by the thiobarbituric acid (TBA; Sigma-Aldrich).
Subsequently, TBA reagent was added to the samples
based on the kit instruction and the final solution was read
by a spectrophotometer (Thermo Fisher Scientific,
Waltham, USA) at 586 nm.26
Statistical analyses. The obtained data were analyzed
by SPSS Software (Version 26.0; IBM Corp., Armonk, USA).
One-way ANOVA test was used for data with normal
distribution and the Kruskal-Wallis was used for data with
abnormal distribution to compare the four experimental
groups. A p-value of less than 0.05 was considered
statistically significant.
Results
Sperm characteristics in fresh and frozen-thawed
semen diluted in different extenders are shown in Table 2.
In fresh diluted semen, curvilinear velocity (VCL), straight-
line velocity (VSL) and average path velocity (VAP) were
significantly higher in Steridyl and AndroMed than in the
manual (p < 0.05; Table 2). Moreover, VSL and VAP were
significantly higher in semen diluted with AndroMed than
Triladyl. The mean angular displacement (MAD) of the
Triladyl, Steridyl, and AndroMed groups was significantly
(p < 0.05) higher than the manual group. The linearity
(LIN) of fresh semen diluted in the AndroMed was
significantly higher than the manual (p < 0.05; Table 2).
The highest progressive motility (PM) and viability of
frozen-thawed semen could be observed in the Triladyl,
which was higher than the manual group (Table 2).
Motility parameters such as VCL, VSL, VAP, amplitude of
lateral head displacement (ALH) and beat-cross frequency
(BCF) with Trilady were higher than in other groups
indicating a difference with the manual group. Plasma
membrane integrity in the Triladyl was higher than in the
manual group (p < 0.05; Table 2). The percentage of DNA
damage with Triladyl and AndroMed was lower than in
the manual group (p < 0.05; Table 2). The percentage of
chromatin integrity in the Triladyl was higher than in the
manual group (p < 0.05; Table 2). The TAC of the Triladyl
and AndroMed groups was higher than the manual group
(p < 0.05), whereas, the MDA of the Triladyl, Steridyl and
AndroMed groups was lower than the manual group
(p < 0.05; Table 2).
376 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379

Table 2. Comparison of sperm quality characteristics for the fresh semen extender in different extenders. Data are presented as the mean
± standard deviation. All the semen quality parameters and blood serum antioxidant enzymes were compared in all groups and classified
based on fresh sperm progressive motility and post thawing.
Parameters Manual Triladyl Steridyl AndroMed
Fresh semen extender
PM (%) 69.78 ± 12.40a 76.61 ± 10.46a 78.21 ± 8.52a 75.21 ± 9.28a
VCL (µm sec-1) 36.68 ± 14.38a 42.22 ± 10.87ab 48.15 ± 12.98b 52.12 ± 15.24b
VSL (µm sec-1) 19.33 ± 8.20a 20.55 ± 6.67 ab 27.41 ± 9.72bc 31.04 ± 10.88b
VAP (µm sec-1) 24.44 ± 9.48a 26.40 ± 6.99 ab 33.08 ± 9.74bc 36.29 ± 11.43b
MAD 26.58 ± 14.72a 34.43 ± 14.96b 33.49 ± 15.29b 34.07 ± 18.67b
ALH (µm) 1.96 ± 0.71a 2.10 ± 0.45a 2.18 ± 0.41a 2.27 ± 0.54a
BCF (Hz) 2.18 ± 1.95a 2.11 ± 1.82a 2.09 ± 1.71a 3.22 ± 7.09a
LIN (%) 42.45 ± 8.98a 43.17 ± 8.57ab 48.39 ± 9.94ab 48.69 ± 8.61b
Viab (%) 82.02 ± 8.03a 83.33 ± 8.21a 82.47 ± 9.55a 81.27 ± 6.37a
Morph (%) 7.87 ± 3.81a 7.50 ± 5.73a 7.97 ± 5.23a 7.19 ± 4.89a
Head (%) 2.67 ± 2.29a 2.93 ± 4.22a 2.63 ± 3.22a 2.59 ± 3.23a
Mid.P (%) 1.50 ± 0.93 a 1.18 ± 0.88 a 1.16 ± 0.78a 1.10 ± 1.01a
Tail (%) 2.51 ± 1.82a 2.47 ± 1.84a 2.80 ± 2.80a 2.54 ± 1.82a
Cy.D (%) 1.17 ± 1.10 a 0.91 ± 1.14 a 1.37 ± 2.25 a 0.95 ± 0.93 a
HOST (%) 57.93 ± 10.89a 61.59 ± 11.52a 62.43 ± 11.24a 64.03 ± 12.23a
Frozen-thawed semen extender
PM (%) 44.91 ± 16.13a 55.69 ± 14.39 b 49.08 ± 12.88ab 52.41 ± 15.67ab
VCL (µm sec ) -1 24.81 ± 11.73 a 32.60 ± 14.43 ab 27.49 ± 10.83 b 40.25 ± 16.92b
VSL (µm sec-1) 10.65 ± 5.12a 13.13 ± 5.49ab 13.74 ± 5.33ab 18.78 ± 8.93b
VAP (µm sec-1) 14.72 ± 6.94a 19.01 ± 7.97ab 17.75 ± 6.91ab 24.54 ± 11.03b
MAD 22.93 ± 11.13a 32.05 ± 14.47ab 22.51 ± 9.28a 34.88 ± 13.24b
ALH (µm) 1.64 ± 0.68a 2.09 ± 0.78ab 1.64 ± 0.60 a 2.28 ± 0.85b
BCF (Hz) 2.32 ± 1.29a 3.29 ± 1.31b 2.33 ± 1.05ab 3.33 ± 1.43b
LIN (%) 24.33 ± 8.80a 29.81 ± 7.21a 29.20 ± 7.56a 30.98 ± 9.82a
Viab (%) 46.18 ± 9.31a 57.59 ± 12.86 b 54.47 ± 12.41b 49.64 ± 12.26ab
Morph (%) 11.15 ± 5.67a 10.16 ± 6.37a 11.17 ± 7.22a 10.69 ± 6.14a
Head (%) 3.65 ± 3.32a 3.27 ± 3.10a 3.44 ± 4.26a 3.14 ± 2.70a
Mid.P (%) 1.63 ± 1.56a 1.24 ± 0.85a 1.61 ± 1.07a 1.48 ± 1.14a
Tail (%) 5.07 ± 3.93a 5.21 ± 5.56a 5.55 ± 5.10a 5.32 ± 4.72a
Cy.D (%) 0.78 ± 0.91a 0.43 ± 0.73a 0.56 ± 0.49a 0.74 ± 0.87a
HOST (%) 34.07 ± 8.39a 41.95 ± 9.15b 37.73 ± 11.12ab 39.07 ± 10.14ab
DNA damage (%) 33.35 ± 3.85a 25.19 ± 3.41b 29.65 ± 6.44a 27.13 ± 4.76b
Chromatin Integrity (%) 2.21 ± 3.53a 1.94 ± 3.19b 2.18 ± 9.15ab 2.07 ± 9.15ab
TAC (mmol L-1) 1.08 ± 0.25a 2.49 ± 0.17b 1.33 ± 0.20a 2.17 ± 0.13b
MDA (nmol mL-1) 6.39 ± 0.22a 2.77 ± 0.35b 5.19 ± 0.41c 3.04 ± 0.39b
PM: progressive motility, VCL: curvilinear velocity, VSL: straight-line velocity, VAP: average path velocity, ALH: amplitude of lateral head
displacement, MAD: mean angular displacement, BCF: beat cross-frequency, LIN: linearity, Viab: viability, Morph: abnormal morphology,
Head: head abnormality, Mid.P: mid-piece abnormality, Tail: tail abnormality, Cy.D: cytoplasmic droplet, and HOST: hypo-osmotic swelling
test, TAC: total antioxidant capacity, MDA: malondialdehyde, VCL: curvilinear velocity, and STR: straightness.
abc Different letters in the same line indicate a significant difference between the groups (p < 0.05).

Discussion The highest parameter of DNA and plasma membrane

Abdel-Aziz Swelum et al. conducted a study in the
University of Saudi Arabia to examine the effectiveness of
Triladyl, Steridyl, AndroMed, SHOTOR and Optixcell for
camel semen maintenance. In the pre-cryopreservation
evaluation, PM was higher with SHOTOR. SHOTOR and
Triladyl had better DNA viability and integrity. Triladyl
resulted in plasma membrane integrity than other
extenders. In the post-thaw evaluation, the PM parameter
was significantly higher in Triladyl. The VAP, VSL, VCL
parameters were higher in Triladyl. The highest amount of
parameter LIN was observed in Triladyl and AndroMed.
integration was obtained with SHOTOR. In the present
study, PM, straightness (STR), LIN, ALH, BCF, VAP, VSL,
and VCL showed better post-thaw evaluation, and the best
performance was recorded in Triladyl. The sperm stored
in Triladyl had more efficient flagellar structures. The best
sperm quality after thawing, the lowest sperm abnormality,
the best DNA integrity, plasma membrane integrity and
increased motility were observed in SHOTOR and Triladyl.
In our study, fresh sperm showed the most PM and
viability of frozen semen in Triladyl and no difference was
observed between the experimental groups in terms of
sperm morphology parameters. The parameters of PM,
 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379
VAP, VSL, VCL, and HOST Triladyl were different from the
other groups concerning frozen semen, while there was no
statistically significant difference between the
experimental groups concerning the LIN parameter. The
results of our research on Simmental Bull were consistent
with the results of Abdel-Aziz Swelum et al.27
Suhardi et al., conducted a study on three factors of
motility, viability and abnormality of Bali bull sperm with
AndroMed and Triladyl egg yolk-tris stored at 4.00 ˚C.28
AndroMed and egg yolks were compared. Sperm motility,
viability and abnormalities were observed and
compared, confirming the superiority of egg yolk-tris to
AndroMed in three parameters sperm motility, viability,
and a lower percentage of abnormalities when stored for
four days at 4.00 ˚C.
In our study, fresh sperm showed the most PM and
viability of frozen semen in Triladyl, while, no significant
difference was observed between the experimental groups
in terms of sperm morphology parameters.28
In a research study in 2015, Chaudhari et al.,
investigated the relative efficacy of egg yolk and soya milk-
based on semen cryopreservation of buffalos (Bubalus
bubalis).15 They evaluated the comparative effects of
ordinary egg yolk (TFYG) and commercial soybean-based
(Bioxcell and Optixcell) on semen cryopreservation of
Bubalus bubalis in terms of sperm motility, viability,
morphology and acrosome integrity. Parameters such as
greater sperm motility and viability and less abnormal
morphology in semen were found in TFYG than in the
Bioxcell in sperm post-cryopreservation examination.
Compared to our study, the highest PM and viability of
frozen semen were observed in Triladyl. Plasma
membrane integrity in the Triladyl was higher than in the
manual group and only the morphology was not different
from the control group. The results obtained by Chaudhari
et al. were consistent with our results.15 Chaudhari et al.
suggested that soy-based Bioxcell had fewer protective
effects on maintaining the integrity of the sperm plasma
membrane and indirectly reflected on sperm motility
which was consistent with our findings.15
In a study of African buffalo elephants by Herold et al.
the effect of equilibrium time of semen diffusers on sperm
quality was investigated after transplantation of bonds
such as motility, longevity and complete acrosome
parameters commonly used for semen.29 The use of
AndroMed resulted in symptoms of much higher
percentages of lost acrosomes than Triladyl and ROFB
groups.21 The results of our research in the three
parameters of motility, survival and acrosome test were
consistent with Herold's et al. research on sperm count
after cryopreservation.
In a study by Fukui et al. at the Animal Reproduction
Laboratory, the fertility of intrauterine ewes with thawed
frozen semen using a soy-based semen (AndroMed) was
compared to intrauterine ewes inoculated with thawed
377
frozen semen using Tris-based extenders containing egg
yolk or bovine serum albumin (BSA). The present results
indicated that a semen enhancer containing egg yolk can
be replaced with the non-animal AndroMed.30
Our study on frozen sperm, the most PM and viability
of frozen semen were observed in the Triladyl, which was
higher than the manual group. Parameters VCL, VSL, VAP,
ALH, and BCF with the Triladyl were higher than the other
groups. Plasma membrane integrity in the Triladyl was
higher than in the manual group.
Another study, conducted by Al-Bulushi et al., Oman
Animal Research and Center, evaluated the quality of
semen in Triladyl, green buffer and Optixcell with fresh
semen.30 Two extenders based on egg yolk (Biladyl or
green buffer and Triladyl) and five extenders based on
synthetic and non-animal compounds (Optixcell, EquiPlus,
INRA96, Bioxcell, AndroMed) were diluted and stored at
4.00 ˚C for 48 hr.
The survival parameters, acrosome integrity and total
sperm motility were then examined for maximum sperm
motility and healthy acrosome after 48 hr of storage with
Triladyl and green buffer. After 48 hr of storage with
Triladyl, the results were consistent with our study
concerning the parameters of viability and motility of fresh
sperm and the acrosome test performed on frozen sperm.31
In a 2012 study at the German Farm Animal Biological
Institute, John Beran and his colleagues ranked extenders
in terms of sperm motility including reductions in sperm
motility in fresh and post-thaw ejaculates. During the
entire thermodynamic test and in the proportion of live
and dead sperm after thawing, the difference in sperm
motility during entire thermodynamic experiment was
significant. Triladyl significantly increased the percentage
of motility and live sperm after melting.8 Ejaculation was
collected once a week from four Holstein bulls. Each of 20
ejaculate samples from each bull was extended with 4
different developing agents AndroMed, Bioxcell without
egg yolk, Triladyl and Optidyl with ionized egg yolk and
used fresh. The results confirmed a significant difference
in the volume, density and activity of sperm as well as in
the share of live and dead sperm after collection. In
reducing sperm motility in fresh ejaculate, after thawing,
as well as in the proportion of live and dead sperm after
thawing, the extenders that are ranked in terms of sperm
motility are Optidyl, Triladyl, AndroMed and Bioxcell,
indicating the higher quality of artificial insemination
doses was produced using egg yolk extenders. The
difference in sperm motility was significant. Egg yolk
expanders after thawing significantly increased the
percentage of viable sperm. The results of John Beran's
research were consistent with our research.8
According to our study in post-cryopreservation
evaluation, PM and survival had better results with
Triladyl. VAP, ALH and BCF parameters were higher with
Triladyl indicating differences with the manual group.
378 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379
The potential predictive parameter for semen
fertilization capacity in bovines is the percentage of PM.7
Triladyl had better performance regarding this important
parameter than the other groups.
Concerning the total anti-oxidant evaluation and lipid
peroxidation, no research has been conducted related to
midwifery and reproductive diseases. The results of this
study could be considered as the first evaluation of the
parameter of total anti-oxidant capacity. Triladyl did not
show a statistical difference with the manual group, while,
MDA of Triladyl, Steridyl, and AndroMed groups was
significantly lower than that of the manual group.
Acknowledgments
This research work has been supported by a research
grant from the Urmia University. We would like to thank
the Vice Chancellor for Research of Urmia University and
authorities in Iran Simmental Cattle Breeding Center
(Amard Dam Tabarestan Company, Amol, Iran).
Conflict of interest
The authors declare no conflict of interest.
References
1. Ramazani N, Mahd Gharebagh F, Soleimanzadeh A, et
al. The influence of L‐proline and fulvic acid on
oxidative stress and semen quality of buffalo bull
semen following cryopreservation. Vet Med Sci 2023;
doi: 10.1002/vms3.1158.
2. Ugur MR, Abdelrahman AS, Evans HC, et al. Advances in
cryopreservation of bull sperm. Front Vet Sci 2019; 6:
268. doi: 10.3389/fvets.2019.00268.
3. Ingermann RL, Holcomb M, Robinson ML, et al. Carbon
dioxide and pH affect sperm motility of white sturgeon
(Acipenser transmontanus). J Exp Biol 2002; 205(Pt
18): 2885-2890.
4. Tejada RI, Mitchell JC, Norman A, et al. A test for the
practical evaluation of male fertility by acridine orange
(AO) fluorescence. Fertil Steril 1984; 42(1): 87-91.
5. Peris-Frau P, Soler AJ, Iniesta-Cuerda M, et al. Sperm
cryodamage in ruminants understanding the molecular
changes induced by the cryopreservation process to
optimize sperm quality. Int J Mol Sci 2020; 21(8): 2781.
6. Manisha WH, Richa R, Deepali J. Oxidative stress and
antioxidants: An overview. IJARR 2017; 2(9): 110-119.
7. Li Y, Kalo D, Zeron Y, et al. Progressive motility - a
potential predictive parameter for semen fertilization
capacity in bovines. Zygote 2014; 24(1): 70-82.
8. Beran J, Stádník L, Bezdíček, J, et al. Effect of sire and
extender on sperm motility and share of live or dead
sperm in bulls’ fresh ejaculate and in AI doses after
thawing. Archiv Tierzucht 2012; 55(3): 207-218.
9.
10. Murphy EM, Murphy C, O'Meara C, et al. A comparison
of semen diluents on the in vitro and in vivo fertility of
liquid bull semen. J Dairy Sci 2017; 100(2): 1541-1554.
11. Raheja N, Choudhary S, Grewal S, et al. A review on
semen extenders and additives used in cattle and
buffalo bull semen preservation. J Entomol Zool Stud
2018; 6(3): 239-245.
12. Sharafi M, Forouzanfar M, Hosseini SM, et al. In vitro
comparison of soybean lecithin based-extender with
commercially available extender for ram semen cryo-
preservation. Int J Fertil Steril 2009; 3(3): 149-152.
13. Malekinejad H, Mirzakhani N, Razi M, et al. Protective
effects of melatonin and Glycyrrhiza glabra extract on
ochratoxin A - - - induced damages on testes in mature
rats. Hum Exp Toxicol 2011; 30(2): 110-123.
14. Contri A, Valorz C, Faustini M, et al. Effect of semen
preparation on casa motility results in cryopreserved
bull spermatozoa. Theriogenology 2010; 74(3): 424-435.
15. Karamfilov S, Nikolov V. First lactation milk production
of cows of the Simmental breed reared in Bulgaria.
Bulg J Agric Sci 2019; 25(2): 363-369.
16. Chaudhari DV, Dhami AJ, Hadiya KK, et al. Relative
efficacy of egg yolk and soya milk-based extenders for
cryopreservation (−196°C) of buffalo semen. Vet World
2015; 8(2): 239-244.
17. Kazerooni T, Asadi N, Jadid L, et al. Evaluation of
sperm’s chromatin quality with acridine orange test
chromomycin A3 and aniline blue staining in couples
with unexplained recurrent abortion. J Assist Reprod
Genet 2009; 26(11-12): 591-596.
18. Campbell RC, Dott HM, Glover TD. Nigrosin eosin as a
stain for differentiating live and dead spermatozoa. J
Agric Sci 1956; 48(1): 1-8. doi: 10.1017/S00218596-
0003029X.
19. Ramu S, Jeyendran RS. The hypo-osmotic swelling test
for the evaluation of sperm membrane integrity.
Methods Mol Biol 2013; 927: 21-25.
20. Revell SG, Mrode RA. An osmotic resistance test for
bovine semen. Anim Reprod Sci 1994; 36(1-2): 77-86.
21. Smirnov V. Granting even more importance to the
problem of ram sperm dilution and storage [Russian].
Ovtsevodstvo 1976; 1: 38-39.
22. El-Saied M, Farag E A, Youssef YE, et al. Sperm
chromatin structure assay in varicocele patients, J Pan-
Arab League Dermatol 2005; 16(1): 141-148.
23. Hosseini S, Qarachorloo M, Ghiasi Tarzi B, et al. A
review of antioxidant capacity assays (reactions,
methods, pros and cons) [Persian]. Food Technol Nutr
(2014);11(4): 89-111.
24. Zargari F. The role of oxidative stress and free radicals
in diseases [Persian]. Razi J Med Sci 2020; 27(2): 10-22.
Draper HH, Hadley M. Malondialdehyde determination
as index of lipid peroxidation. Methods Enzymol J
1990; 186: 421-430.
25. Gundogan M, Yeni D, Avdatek F, et al. Influence of
 K. Maleki et al. Veterinary Research Forum. 2023; 14 (7) 373 - 379
sperm concentration on the motility, morphology,
membrane and DNA integrity along with oxidative
stress parameters of ram sperm during liquid storage.
Anim Reprod Sci 2010; 122(3-4): 200-207.
26. Ramazani N, Mahd Gharebagh F, Soleimanzadeh A.
Reducing oxidative stress by κ-carrageenan and
C60HyFn: The post-thaw quality and antioxidant
status of Azari water buffalo bull semen. Cryobiology
2023; S0011-2240(23)00032-9. doi: 10.1016/j.
cryobiol.2023.04.003.
27. Abdel-Aziz Swelum A, Saadeldin IM, Ba-Awadh H, et al.
Efficiency of commercial egg yolk-free and egg yolk-
supplemented tris-based extenders for dromedary
camel semen cryopreservation. Animals (Basel) 2019;
9(11): 999. doi: 10.3390/ani9110999.
379
28. Suhardi R, Megawati N, Ardhani F, et al. Motility, viability,
and abnormality of the spermatozoa of Bali bull with
Andromed® and egg yolk-tris extenders stored at 4 °C.
Iran J Appl Anim Sci 2020; 10(2): 249-256.
29. Herold FC, Dehaas K, Cooper D, et al. Comparison of
three different media for freezing of epididymal sperm
from the African buffalo (Syncerus caffer) and influence
of equilibration time on the post-thaw sperm quality.
Onderstepoort J Vet Res 2004; 71(3): 203-210.
30. Fukui Y, Kohno H, Togari T, et al. Fertility after artificial
insemination using a soybean-based semen extender in
sheep. J. Reprod Dev 2008; 54(4): 286-289.
31. Al-Bulushi S, Manjunatha BM, Bathgate R, et al. Liquid
storage of dromedary camel semen in different
extenders. Anim Reprod Sci 2019; 207: 95-106.